A label-free real-time method for measuring glucose uptake kinetics in yeast.
Michaelis–Menten constant
glucose uptake assay
hexose transporters
hxt0 yeast
pHluorin
radiolabeled glucose
Journal
FEMS yeast research
ISSN: 1567-1364
Titre abrégé: FEMS Yeast Res
Pays: England
ID NLM: 101085384
Informations de publication
Date de publication:
16 01 2021
16 01 2021
Historique:
received:
19
10
2020
accepted:
16
12
2020
pubmed:
19
12
2020
medline:
5
11
2021
entrez:
18
12
2020
Statut:
ppublish
Résumé
Glucose uptake assays commonly rely on the isotope-labeled sugar, which is associated with radioactive waste and exposure of the experimenter to radiation. Here, we show that the rapid decrease of the cytosolic pH after a glucose pulse to starved Saccharomyces cerevisiae cells is dependent on the rate of sugar uptake and can be used to determine the kinetic parameters of sugar transporters. The pH-sensitive green fluorescent protein variant pHluorin is employed as a genetically encoded biosensor to measure the rate of acidification as a proxy of transport velocity in real time. The measurements are performed in the hexose transporter-deficient (hxt0) strain EBY.VW4000 that has been previously used to characterize a plethora of sugar transporters from various organisms. Therefore, this method provides an isotope-free, fluorometric approach for kinetic characterization of hexose transporters in a well-established yeast expression system.
Identifiants
pubmed: 33338229
pii: 6041724
doi: 10.1093/femsyr/foaa069
pmc: PMC7811510
pii:
doi:
Substances chimiques
Green Fluorescent Proteins
147336-22-9
Glucose
IY9XDZ35W2
Types de publication
Journal Article
Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Subventions
Organisme : NIGMS NIH HHS
ID : R01 GM123103
Pays : United States
Informations de copyright
© The Author(s) 2020. Published by Oxford University Press on behalf of FEMS.
Références
Front Chem. 2018 May 25;6:183
pubmed: 29888221
ACS Synth Biol. 2015 Sep 18;4(9):975-86
pubmed: 25871405
Microb Cell Fact. 2016 Jul 25;15(1):127
pubmed: 27455954
PLoS One. 2015 Mar 27;10(3):e0121985
pubmed: 25816250
Yeast. 1994 Dec;10(12):1553-8
pubmed: 7725790
J Gen Microbiol. 1989 Sep;135(9):2413-22
pubmed: 2697747
Yeast. 2001 Dec;18(16):1515-24
pubmed: 11748728
Nat Protoc. 2007;2(1):1-4
pubmed: 17401330
Folia Microbiol (Praha). 1994;39(6):557-9
pubmed: 8550023
Biotechnol Biofuels. 2012 Mar 16;5:14
pubmed: 22424089
Am J Physiol. 1989 May;256(5 Pt 2):F957-64
pubmed: 2719125
J Bacteriol. 1994 Feb;176(4):953-8
pubmed: 8106337
Biochim Biophys Acta. 2011 Oct;1810(10):933-44
pubmed: 21421024
FEMS Yeast Res. 2002 Aug;2(3):283-91
pubmed: 12702277
Nature. 1998 Jul 9;394(6689):192-5
pubmed: 9671304
Anal Biochem. 1976 May 7;72:248-54
pubmed: 942051
Anal Biochem. 2016 Jul 15;505:43-50
pubmed: 27130501
Sci Rep. 2018 Aug 10;8(1):11985
pubmed: 30097598
Eur J Biochem. 1997 Apr 15;245(2):324-33
pubmed: 9151960
Nature. 2020 Feb;578(7794):321-325
pubmed: 31996846
Yeast. 1996 Apr;12(5):439-47
pubmed: 8740417
Biochim Biophys Acta. 2002 Aug 15;1572(1):143-8
pubmed: 12204343
Microbiology (Reading). 2009 Jan;155(Pt 1):268-278
pubmed: 19118367
Cell Microbiol. 2007 Apr;9(4):1004-13
pubmed: 17381432
Am J Physiol. 1992 Jul;263(1 Pt 2):H276-84
pubmed: 1636765
Curr Protoc Pharmacol. 2011 Dec;Chapter 12:Unit 12.14.1-22
pubmed: 22147347
Biochem J. 2001 May 1;355(Pt 3):625-31
pubmed: 11311123
Nucleic Acids Res. 1997 Jan 15;25(2):451-2
pubmed: 9016579
Methods Mol Biol. 2018;1713:123-135
pubmed: 29218522
Proc Natl Acad Sci U S A. 2014 Apr 8;111(14):5159-64
pubmed: 24706835
Sci Total Environ. 2019 Jun 1;667:464-474
pubmed: 30833245
FEBS Lett. 1999 Dec 31;464(3):123-8
pubmed: 10618490